scholarly journals Correlative immunofluorescence and electron microscopy on the same section of epon-embedded material.

1985 ◽  
Vol 33 (2) ◽  
pp. 165-171 ◽  
Author(s):  
C L Rieder ◽  
S S Bowser
Keyword(s):  
Electron Microscopy ◽  
Core Protein ◽  
Fluorescein Isothiocyanate ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Immunofluorescent Localization ◽  
Cellular Inclusions ◽  
Tumor Virus ◽  
Specific Antigens ◽  
First Time

Semithick (0.25-0.50 micron) sections, cut from cells stained with fluorescein isothiocyanate (FITC)-conjugated antibodies prior to embedding in Epon, show high resolution patterns of immunofluorescence against a background void of autofluorescence. These same sections can then be viewed, after uranyl and lead staining, in the electron microscope. We clearly establish the specificity of this same-section correlative immunofluorescence-electron microscopy approach by showing that the immunofluorescent patterns observed in such sections of cells, stained prior to embedding for the indirect immunofluorescent localization of tubulin, follows the distribution of microtubules within the same sections as determined by electron microscopy. We then use this method to demonstrate for the first time that the 57 kD core protein of wound tumor virus is associated, at the ultrastructural level, with two distinct cellular inclusions in virally infected AC-20 cells. In some instances the fidelity in the correlation between the distribution of immunofluorescently labeled antigens and the ultrastructure in the same section eliminates the need to employ more complex procedures for labeling antigens for ultrastructural detection. This technique, therefore, provides a rapid and simple first approach to many problems that require the ultrastructural localization of specific antigens.

scholarly journals Visualization of anti-sperm plasma membrane IgG and Fab as a method for localization of boar sperm membrane antigens.

1982 ◽  
Vol 30 (12) ◽  
pp. 1217-1227 ◽  
Author(s):  
L D Russell ◽  
R N Peterson ◽  
T A Russell
Keyword(s):  
Plasma Membrane ◽  
Protein A ◽  
Fluorescein Isothiocyanate ◽  
Ultrastructural Localization ◽  
Surface Antigens ◽  
Ultrastructural Level ◽  
Simple Method ◽  
Sperm Surface ◽  
Sperm Plasma Membrane ◽  
Sperm Antigens

A simple method for ultrastructural localization of sperm surface antigens by direct visualization of bound antibodies is presented. Anti-sperm plasma membrane (ASPM) immunoglobulin (Ig) G, visualized in tissues treated with an osmium:ferrocyanide mixture, projected 11-13 nm from the surface and ASPM Fab fragments projected 8-10 nm from the surface. The density of IgG labeling, as subjectively estimated, corresponded to indirect immune fluorescein isothiocyanate, indirect immunoferritin, and sperm-vesicle labeling patterns. Agglutination of sperm vesicles and sperm were demonstrated and the linking antibody visualized. A second antibody on protein A directed against ASPM IgG made the immunologic tag more apparent and indicated, in disrupted sperm preparations, labeling of both sides of the plasma membrane. The method provides for easy and sensitive localization of sperm surface antigens at the ultrastructural level and is presently being used to localize specific sperm antigens.

Ultrastructural localization of succinate dehydrogenase in a self-parasitic isolate of Saprolegnia megasperma

1977 ◽  
Vol 23 (5) ◽  
pp. 491-496
Author(s):  
Sharon Faye Murrin ◽  
Richard A. Nolan
Keyword(s):  
Electron Microscopy ◽  
Succinate Dehydrogenase ◽  
Cell Walls ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Mitochondrial Membranes ◽  
Hyphal Cell ◽  
Dense Deposits

The enzyme succinate dehydrogenase (SDH, succinate: (acceptor) oxidoreductase, EC 1.3.99.1) was localized by the combined techniques of cytochemistry and electron microscopy in the hyphae of a self-parasitizing isolate of Saprolegnia megasperma Coker. The enzyme was localized in the mitochondrial membranes; its activity was inhibited by malonate. Electron-dense deposits, whose formation was not prevented by the addition of malonate, appeared outside of the hyphal cell walls. No evidence was found at the ultrastructural level within the vegetative hyphae for any abnormalities which could be linked to the phenomenon of self-parasitism.

The energy filtering TEM (EFTEM) in modern biological transmisson Electron Microscopy

1995 ◽  
Vol 53 ◽  
pp. 668-669
Author(s):  
W. Probst ◽  
V.E. Bayer
Keyword(s):  
Electron Microscopy ◽  
Chemical Elements ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Limited Information ◽  
Energy Filtering ◽  
Transmission Electron ◽  
New Findings ◽  
Biochemical Analyses ◽  
Selection Of

Modern biological electron microscopy can no longer be a static tool merely describing morphology. In addition to ultrastructural information, insights into the molecular and chemical composition of a sample are needed so that new findings stemming from molecular biological and biochemical analyses can be given meaning in an ultrastructural context. Biological electron microscopy will be an essential tool for future discoveries involving the ultrastructural localization of molecules and chemical elements, and it will provide a means to identify the ultrastructural basis for a variety of reaction mechanisms. Many messenger compounds are currently known which can produce dynamic changes of either a subtle or dramatic nature at the ultrastructural level, but only the most basic of these can be examined using a conventional transmission electron microscope (CTEM). CTEMs provide limited information because they perform conventional imaging and do not employ all the signals available for analysis. Unlike a CTEM, an EFTEM permits the selection of a defined energy (wavelength) of electrons which are then used for imaging.

scholarly journals Application of the FITC-anti-FITC-gold system to ultrastructural localization of antigens.

1991 ◽  
Vol 39 (12) ◽  
pp. 1725-1728 ◽  
Author(s):  
G J van Dam ◽  
B J Bogitsh ◽  
J A Fransen ◽  
D Kornelis ◽  
R J van Zeyl ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Fluorescein Isothiocyanate ◽  
High Specificity ◽  
Ultrastructural Localization ◽  
Good Alternative ◽  
Ultrastructural Level ◽  
Electron Microscopic ◽  
Protein Conjugates ◽  
Specificity And Sensitivity ◽  
Microscopic Studies

We report the application of a fluorescein isothiocyanate (FITC)-anti-FITC method to localize antigens at the ultrastructural level. In the systems studied, the anti-FITC-based detection method displays high specificity and sensitivity. These observations, combined with ease of production and with availability of FITC-protein conjugates, suggest that the FITC-anti-FITC method is a good alternative to presently used methods and is widely applicable to immunochemical and immunocytochemical procedures. The same preparation and protocol can be used for light and electron microscopic studies, thereby reducing possible artifacts introduced if different procedures are used. In the present study, two systems were used to test the method. One system used an FITC-labeled monoclonal antibody (MAb) to schistosome circulating cathodic antigen. In this system, the label was detected in the gut of adult Schistosoma mansoni by an anti-FITC MAb conjugated to 10-nm gold particles. The second system used human IgM antibodies pooled from patients infected with Schistosoma mansoni. In this system detection was accomplished using an anti-human IgM-FITC conjugate followed by the anti-FITC-Au antibody conjugate.

Permeability of Endometrial Blood Vessels to Exogenous Horse Radish Peroxidase in Mice. Electron Microscope Study

1973 ◽  
Vol 31 ◽  
pp. 562-563
Author(s):  
M.C. Castillo-Jessen ◽  
A. González-Angulo
Keyword(s):  
Electron Microscopy ◽  
Blood Vessels ◽  
Electron Microscope Study ◽  
Ultrastructural Level ◽  
Low Molecular Weight ◽  
Horse Radish Peroxidase ◽  
Intravascular Injection ◽  
Normal Morphology ◽  
Functional Implications ◽  
Low Molecular Weight Proteins

Information regarding the normal morphology of uterine blood vessels at ultrastructural level in mammals is scarce Electron microscopy studies dealing with endometrial vasculature despite the functional implications due to hormone priming are not available. Light microscopy observations with combined injection of dyes and microradiography along with histochemical studies does not enable us to know the detailed fine structure of the possible various types of blood vessels in this tissue. The present work has been designed to characterize the blood vessels of endometrium of mice as well as the behavior of the endothelium to injection of low molecular weight proteins during the normal estrous cycle in this animal. One hundred and forty female albino mice were sacrificed after intravascular injection of horse radish peroxidase (HRP) at 30 seconds, 5, 15, 30 and 60 minutes.

Immunocytochemistry. A Review of Methodology for Electron Microscopic Applicaiton

1974 ◽  
Vol 32 ◽  
pp. 328-329
Author(s):  
Joseph E. Mazurkiewicz
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Ultrastructural Level ◽  
Electron Microscopic ◽  
Biological Interest ◽  
Investigative Approach ◽  
Immunologic Activity ◽  
Dense Label

Immunocytochemistry is a powerful investigative approach in which one of the most exacting examples of specificity, that of the reaction of an antibody with its antigen, isused to localize tissue and cell specific molecules in situ. Following the introduction of fluorescent labeled antibodies in T950, a large number of molecules of biological interest had been studied with light microscopy, especially antigens involved in the pathogenesis of some diseases. However, with advances in electron microscopy, newer methods were needed which could reveal these reactions at the ultrastructural level. An electron dense label that could be coupled to an antibody without the loss of immunologic activity was desired.

Erythrophagocytosis by Entamoeba histolytica.- Ultrastructural Study

1972 ◽  
Vol 30 ◽  
pp. 156-157 ◽  
Author(s):  
Norberto Treviño ◽  
Alfredo Feria-Velasco ◽  
I. Ruiz de Chávez
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Entamoeba Histolytica ◽  
Ultrastructural Study ◽  
Saline Solution ◽  
Physiological Saline ◽  
Ultrastructural Level ◽  
Hot Plate ◽  
Physiological Saline Solution ◽  
Axenic Conditions

Although erythrophagocytosis by various species of Entamoeba is a well known phenomenon this has not yet been studied in detail at the ultrastructural level. The present work deals with the description of the incorporation process of erythrocytes by trophozoites of E. histolytica. For this study, trophozoites of E. histolytica, HK-9:NIH strain cultured in axenic conditions and washed human erythrocytes were placed on a hot plate at 37°C in physiological saline solution. After 5 minutes, 2.5% glutarldehyde was added and the samples were processed according to conventional techniques for electron microscopy.Based upon light microscopy studies on living trophozoites in contact with erythrocytes, it seems that erythrophagocytosis only takes place in one pole of the parasite.

Comparative strengths and weaknesses of peroxidase and colloidal gold in pre- and post-embedding immunolabeling techniques

1987 ◽  
Vol 45 ◽  
pp. 1002-1005
Author(s):  
D. R. Abrahamson ◽  
P. L. St.John ◽  
E. W. Perry
Keyword(s):  
Electron Microscopy ◽  
Horseradish Peroxidase ◽  
Light Microscopy ◽  
Colloidal Gold ◽  
Light Microscope ◽  
Ultrastructural Localization ◽  
The Other ◽  
Quantitative Studies ◽  
Dense Suspension ◽  
Antigen Localization

Antibodies coupled to tracers for electron microscopy have been instrumental in the ultrastructural localization of antigens within cells and tissues. Among the most popular tracers are horseradish peroxidase (HRP), an enzyme that yields an osmiophilic reaction product, and colloidal gold, an electron dense suspension of particles. Some advantages of IgG-HRP conjugates are that they are readily synthesized, relatively small, and the immunolabeling obtained in a given experiment can be evaluated in the light microscope. In contrast, colloidal gold conjugates are available in different size ranges and multiple labeling as well as quantitative studies can therefore be undertaken through particle counting. On the other hand, gold conjugates are generally larger than those of HRP but usually can not be visualized with light microscopy. Concern has been raised, however, that HRP reaction product, which is exquisitely sensitive when generated properly, may in some cases distribute to sites distant from the original binding of the conjugate and therefore result in spurious antigen localization.

Ultrastructural Localization of Antigens by Capillary Action Immunocytochemistry

1996 ◽  
Vol 54 ◽  
pp. 40-41
Author(s):  
László G. Kömüves
Keyword(s):  
San Francisco ◽  
Colloidal Gold ◽  
Specific Binding ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Uranyl Acetate ◽  
Adhesive Tape ◽  
Distilled Water ◽  
Capillary Action ◽  
Rabbit Igg

Light microscopic immunohistochemistry based on the principle of capillary action staining is a widely used method to localize antigens. Capillary action immunostaining, however, has not been tested or applied to detect antigens at the ultrastructural level. The aim of this work was to establish a capillary action staining method for localization of intracellular antigens, using colloidal gold probes.Post-embedding capillary action immunocytochemistry was used to detect maternal IgG in the small intestine of newborn suckling piglets. Pieces of the jejunum of newborn piglets suckled for 12 h were fixed and embedded into LR White resin. Sections on nickel grids were secured on a capillary action glass slide (100 μm wide capillary gap, Bio-Tek Solutions, Santa Barbara CA, distributed by CMS, Houston, TX) by double sided adhesive tape. Immunolabeling was performed by applying reagents over the grids using capillary action and removing reagents by blotting on filter paper. Reagents for capillary action staining were from Biomeda (Foster City, CA). The following steps were performed: 1) wet the surface of the sections with automation buffer twice, 5 min each; 2) block non-specific binding sites with tissue conditioner, 10 min; 3) apply first antibody (affinity-purified rabbit anti-porcine IgG, Sigma Chem. Co., St. Louis, MO), diluted in probe diluent, 1 hour; 4) wash with automation buffer three times, 5 min each; 5) apply gold probe (goat anti-rabbit IgG conjugated to 10 nm colloidal gold, Zymed Laboratories, South San Francisco, CA) diluted in probe diluent, 30 min; 6) wash with automation buffer three times, 5 min each; 7) post-fix with 5% glutaraldehyde in PBS for 10 min; 8) wash with PBS twice, 5 min each; 9) contrast with 1% OSO4 in PBS for 15 min; 10) wash with PBS followed by distilled water for5 min each; 11) stain with 2% uranyl acetate for 10 min; 12) stain with lead citrate for 2 min; 13) wash with distilled water three times, 1 min each. The glass slides were separated, and the grids were air-dried, then removed from the adhesive tape. The following controls were used to ensure the specificity of labeling: i) omission of the first antibody; ii) normal rabbit IgG in lieu of first antibody; iii) rabbit anti-porcine IgG absorbed with porcine IgG.

SCANNING ELECTRON MICROSCOPE: POSSIBILITIES FOR STUDYING KIDNEY DISEASES

2020 ◽  
pp. 176-182
Author(s):  
Mamaeva S.N. ◽  
Vinokurov R.R. ◽  
Munkhalova Ya.A. ◽  
Dyakonova D.P. ◽  
Platonova V.A. ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Electron Microscope ◽  
Scanning Electron Microscope ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Filtration Rate ◽  
Kidney Diseases ◽  
First Time ◽  
Scanning Electron

Currently, due to the intensive development of high-tech science-intensive medical and research devices, more and more attention is paid to the development of diagnostics of rare and difficult to diagnose diseases. It is known that among numerous nephropathies, hematuria may be the only symptom of kidney and urinary tract diseases, which complicates their diagnosis and treatment. In order to develop new approaches for the diagnosis of nephropathies, the authors have been studying the morphology of red blood cells in the blood and urine of children and adults using a scanning electron microscope for several years. The paper presents the results of studies of children with various kidney diseases, including IgA-nephropathy, and chronic glomerulonephritis. Scanning electron microscopy was used for the first time to detect nanoparticles on the surface of red blood cells, the size of which is comparable to the size of viruses, which became the basis for one of the authors ' assumptions, namely, the possible transport of certain types of viruses by red blood cells. Thus, some kidney diseases could be considered virus-associated. This paper presents for the first time the results of determining the glomerular filtration rate of both kidneys separately in the study of separate kidney function and of the study of urine smears obtained during catheterization of the ureters in patients with hydronephrosis of one of the kidneys by scanning electron microscopy. As in previous studies, nanoparticles were found on the surface of red blood cells, which leads to the conclusion about the possible viral nature of the disease of the considered patient. In addition, smear images obtained using a microscope showed a significant difference in the elements of the right and left kidneys urine, which did not contradict the data on the study of glomerular filtration rate. According to the authors, the capabilities of the scanning electron microscope can be applied in fundamental research of kidney diseases at the cellular and molecular levels, forming new ideas about their origin, as well as on the basis of which new methods of non-invasive diagnostics can be built.

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